The Na-K-CI and K-CI co-transporters play vital roles in transepithelial salt transport and cellular volume and electrolyte balance. The Na-K-CI co-transporter is a key element in the process of NaCI reabsorption across the mammalian renal epithelium in the thick ascending limb of the loop of Henle (TAL), where it is the site of action of diuretics such as furosemide. The K-CI co-transporter has been proposed to be involved in Na CI reabsorption in the proximal tubule and TAL as well as in K secretion in the distal tubule, but lacking specific inhibitors and molecular probes, it has until now been impossible to confirm these roles. Within the last several years, cDNAs encoding the Na-K-CI co-transporter (NKCC1 and NKCC2) and K-CI co-transporter (KCC1) have been cloned in this laboratory, enabling molecular examination of the transport process. The goal of this project is to understand the mechanisms that underly the function of the Na-K-CI and K-CI co-transporters in the mammalian kidney, and to understand the significance of the co-transporters to overall renal function. The proposed studies will be carried out with knockout mouse models of NKCC2 and KCC1, as well as with rabbit tissues, and with recombinant NKCC and KCC protein expressed in tissue culture cells. Specifically: 1) Structure-function relationships in the renal Na-K-CI co- transporter (NKCC2) will be addressed, examining four hypotheses: that NKCC2b in the macula densa is central to tubuloglomerular feedback; that the three splice variants of NKCC2 convey different ion affinities and that these differences are functionally significant; and that NKCC2 is regulated by direct phosphorylation. 2) The domain of the co-transporter protein that directs apical/basolateral sorting in polarized epithelia will be determined by expression of chimeric NKCC1/NKCC2 constructs in an epithelial cell line. 3) The renal distribution of KCC1 will be determined, and the functional significance of the co-transporter will be assessed in a knockout mouse model.